Hyaluronic acid anti-adhesion barrier

ABSTRACT

Methods of forming crosslinked hyaluronic acid anti-adhesion barriers, crosslinked hyaluronic acid anti-adhesions barriers, methods for preventing or inhibiting adhesions, and methods of promoting healing of a wound are provided. The method of forming the crosslinked hyaluronic acid anti-adhesion barrier includes freeze-drying a solution including hyaluronic acid to form a hyaluronic acid foam, which is then reacted with a crosslinking agent to form a crosslinked hyaluronic acid foam. The crosslinked hyaluronic acid foam is mixed with a solution containing hyaluronic acid to form an anti-adhesion barrier.

BACKGROUND

[0001] 1. Technical Field

[0002] The present disclosure relates to a method of preparing ananti-adhesion barrier from hyaluronic acid, to the anti-adhesion barrierformed thereby, and to methods of using the anti-adhesion barrier.

[0003] 2. Background of the Related Art

[0004] Hyaluronic acid is a viscous mucopolysaccharide found in animaland human tissues such as the umbilical cord, vitreous humor, synovialfluid, blood vessel walls and other connective tissues. Thepolysaccharide consists of repeating disaccharide units made ofalternating D-glucuronic acid and N-acetyl-D-glucosamine residues, andpossesses a molecular weight ranging from about 40,000 to 8,000,000depending on the source and methods of extraction. Hyaluronic acid isfound in between cells complexed with proteins, and forms a jelly due toits ability to retain water. It plays an important role in variousbiological processes, such as cell migration, lubrication, moistening oftissues, and maintenance of cell morphology (Meyers, Physiol. Rev., 27:335, 1947).

[0005] Its natural occurrence in the body and its ability to retainwater have lead to the development and use of hyaluronic acid forvarious therapeutic applications, e.g., the treatment of arthritis, theuse of hyaluronic acid as a vitreous humor substitute, the prevention orinhibition of adhesions following surgery, and the protection of woundsduring healing. However, upon administration to an individual,hyaluronic acid undergoes enzymatic degradation by various enzymes,e.g., hyaluronidase, glucoronidase, and glucosidase, or non-enzymaticdegradation (Pigman et al., Arthritis Rheumatism 4: 240, 1961), and thusdoes not maintain its original viscosity or desired residence time invivo.

[0006] One successful approach to delay degradation of hyaluronic acidwhen administered to the body, and thus preserve its original viscosityand residence time in vivo, has been to modify hyaluronic acid with acrosslinking agent. Various crosslinking agents and methods have beenutilized to crosslink hyaluronic acid for use in various therapeuticapplications. For example, U.S. Pat. No. 4,716,224 describes thecrosslinking of hyaluronic acid with the use of poly-functional epoxycompounds wherein the hyaluronic acid is dissolved and reacted with theepoxy compounds in an alkaline medium. The crosslinked hyaluronic acidis described as useful in the treatment of arthritis and as aningredient of cosmetics.

[0007] U.S. Pat. No. 4,886,787 describes crosslinking hyaluronic acidwith di or polyfunctional epoxides wherein the hyaluronic acid isdissolved and reacted with the epoxides in an acidic solution, in thepresence of an acidic catalyst. The crosslinked hyaluronic acid isproposed to be useful in the treatment of arthritis, as a drug deliveryvehicle, to reduce post-operative adhesions, to promote wound healing,and as a component of cosmetics.

[0008] U.S. Pat. Nos. 4,582,865, 4,605,691 and 4,636,524 describe thereaction of divinyl sulfone as crosslinking agent with hyaluronic acidin an aqueous alkaline solution. The crosslinked hyaluronic acid isdescribed as being useful in cosmetic formulations and drug deliverysystems.

[0009] U.S. Pat. No. 5,356,883 describes crosslinking hyaluronic acidwith the crosslinking agent carbodiimides to produce hydrogels which arepurportedly useful as biocompatible gels, films or sponges.

[0010] U.S. Pat. No. 4,957,744 describes crosslinking hyaluronic acidwith polyhydric alcohols to produce crosslinked hyaluronic acid esters,which are described as useful for treatment of arthritis, and ascomponents of cosmetics. The crosslinking reaction is effected bydissolving and reacting hyaluronic acid with a crosslinking agent inpolar and non-polar solvents.

[0011] U.S. Pat. No. 5,690,961 describes crosslinking hyaluronic acidwith di- or polyanhydrides in a polar, aprotic solvent. Theanhydride-crosslinked hyaluronic acid is proposed to be useful in thetreatment of arthritis, as a drug delivery vehicle, to reduce theformation of post-operative adhesions, to promote wound healing and asan ingredient in cosmetics.

[0012] U.S. Pat. No. 5,532,221 describes the production ofionically-crosslinked hyaluronic acid, wherein an aqueous solution ofhyaluronic acid is contacted with an aqueous polycation solution. Theionically-crosslinked hyaluronic acid is described as useful inpreventing post-operative adhesions.

[0013] In the literature described above, regardless of the particularcrosslinking agent used, the crosslinking of hyaluronic acid is carriedout in a homogenous solution state, wherein hyaluronic acid is dissolvedand reacted with the crosslinking agent in a solution. Since thesolubility of hyaluronic acid in aqueous solution is very low, thereactions carried out in such homogenous solutions are not efficient.Accordingly, recovery of the reaction product can be difficult.

SUMMARY

[0014] A method of forming an anti-adhesion barrier is provided whichcomprises freeze-drying a solution including hyaluronic acid to form afoam, reacting the foam with a crosslinking agent to form a crosslinkedfoam and mixing the crosslinked foam with an aqueous solution containinghyaluronic acid to form the anti-adhesion barrier.

[0015] In another aspect, an anti-adhesion barrier is provided which isa gel produced by combining a freeze-dried crosslinked hyaluronic acidfoam and an aqueous solution including hyaluronic acid.

[0016] In yet another aspect, a two-part kit is provided which comprisesa first part including freeze-dried crosslinked hyaluronic acid foam,and a second part including a solution including hyaluronic acid.

[0017] As stated previously, prior art methods for preparing crosslinkedhyaluronic acid, in general, involve a crosslinking step which isperformed in a homogenous solution state, i.e., hyaluronic acid isdissolved and reacted with a crosslinking agent in a solution. Incontrast, the present method of preparing an anti-adhesion barrierinvolves a unique solid-state crosslinking reaction wherein hyaluronicacid is prepared as a solid, i.e., freeze-dried foam, which is thenreacted with a neat liquid crosslinking agent to yield a crosslinkedhyaluronic acid foam. This method eliminates the concern of lowsolubility of hyaluronic acid in various solvents. Also, because thecrosslinking agents only react with those functional groups accessibleon surfaces, the use of a freeze-dried hyaluronic acid foam provides arelatively large surface area containing sites at which crosslinking canoccur.

[0018] The production of crosslinked hyaluronic acid foam in accordancewith the method of the present invention results in a near-quantitativerecovery of crosslinked hyaluronic acid foam. The excess crosslinkingagents and any reaction by-products can be easily removed by simplewashing. In contrast, recovery of crosslinked hyaluronic acid producedby the methods of the prior art wherein crosslinking is conducted inhomogenous solution state is less efficient and removal of excesscrosslinking agents and reaction by-products is more time consuming.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is an example of a needleless syringe that can be utilizedto mix hyaluronic acid foam with an aqueous solution containinghyaluronic acid to form the anti-adhesion barrier.

[0020]FIGS. 2A and B shows two needleless syringes and a luer lock thatcan be used to mix an aqueous solution containing hyaluronic acid andcrosslinked hyaluronic acid foam to form an anti-adhesion barrier. FIG.2A shows two separate syringes containing crosslinked hyaluronic acidfoam, and an aqueous solution containing hyaluronic acid, and a luerlock which is used for coupling the two syringes. FIG. 2B shows thesyringes coupled via a luer lock. The liquid and solid components aremixed by pushing the plungers back and forth to form a gel. The gel iscollected in one of the syringes prior to its application to a woundsite.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0021] The presently disclosed method of forming an anti-adhesionbarrier includes a novel crosslinking step wherein a freeze-driedhyaluronic acid foam is reacted with a crosslinking agent to produce acrosslinked hyaluronic acid foam, which is then mixed with an aqueoussolution comprising hyaluronic acid to form a hydrogel anti-adhesionbarrier.

[0022] As used herein, the term “hyaluronic acid” refers to hyaluronicacid and its salts such as the sodium, potassium, calcium or magnesium,etc., salts. To form an anti-adhesion barrier as set forth in thepresently claimed method, hyaluronic acid or a biocompatible saltthereof, e.g., sodium, potassium, calcium or magnesium, is dissolved inan aqueous solution, which is preferably pyrogen-free. The molecularweight of hyaluronic acid may range from about 40,000 to 8,000,000depending on the source and the purification methods utilized. Suitablesources of hyaluronic acid include, but are not limited to, umbilicalcord, rooster comb, bacteria, etc. For example, U.S. Pat. No. 4,141,973,the contents of which are incorporated by reference herein describes amethod for extracting hyaluronic acid from rooster combs, and U.S. Pat.No. 4,517,295, the contents of which are incorporated by referenceherein describes a fermentation method for preparing hyaluronic acid.Hyaluronic acid is also commercially available, e.g., from GenzymeCorp., Cambridge, Mass. and Sigma Chemical Co., St. Louis, Mo.

[0023] The molecular weight of hyaluronic acid affects the amount ofhyaluronic acid that is required to achieve gel formation. Accordingly,the concentration of hyaluronic acid used to form a foam in the presentmethod will be adjusted according to the molecular weight of thehyaluronic acid utilized. For example, when using a higher molecularweight hyaluronic acid, a lower concentration of hyaluronic acid isrequired to achieve gel formation. Typically, when using hyaluronic acidhaving a molecular weight of about 1×10⁶ to about 2×10⁶, the solutionincluding hyaluronic acid will contain from about 0.5 to about 4.0% byweight hyaluronic acid.

[0024] The solution including hyaluronic acid is then frozen, preferablyflash-frozen at a temperature of from about −94 to about −130° C. by,e.g., immersing a flask containing the solution including hyaluronicacid in an acetone-dry ice bath. By flash-freezing the solutionincluding hyaluronic acid, a porous hyaluronic acid foam can be obtainedupon freeze-drying the frozen solution including hyaluronic acid.Formation of a foam increases the surface area that is exposed tocrosslinking reagent. The formation of a porous foam allows crosslinkingagent to reach all the surface area. The frozen hyaluronic acid solutionis freeze-dried, preferably at a temperature of from about 0 to about−10° C. and at a pressure of from about 50×10⁻³ to about 1×10⁻³ torr toform a porous foam.

[0025] Subsequently the foam is reacted with a liquid crosslinking agentto produce crosslinked foam. Suitable crosslinking agents are well-knownin the art, and include but are not limited to, di-epoxides,poly-functional epoxides, diisocyanates, polyisocyanates, polyhydricalcohols, water-soluble carbodiimides, diamines, diaminoalkanes,polycarboxylic acids, diacid halides, and dimethylol urea. Preferably,the crosslinking agent is a diacid halide which includes, but is notlimited to, diglycolyl chloride, adipoyl chloride, and sebacyl chloride.More preferably, the diacid halide is diglycolyl chloride. Thecrosslinking agent can be a liquid or rendered liquid, if necessary bydissolving the crosslinking agent in a suitable solvent, e.g., methylenechloride, or N,N-dimethylformamide. When a solvent is used, theconcentration of crosslinking agent is from about 1.0 to about 99.0% byweight, preferably from about 10 to about 80 weight percent of thesolution. It is preferred in the practice of the present invention toemploy substantially pure crosslinking agents.

[0026] To effect crosslinking, the foam is immersed in the liquidcrosslinking agent and stirred for an appropriate period of time,usually for about 24 to about 48 hours. The reaction is usuallyperformed at room temperature, i.e., at about 23° C., however, thetemperature at which the crosslinking reaction is conducted is notcritical.

[0027] The ratio of foam to crosslinking agent is from about 3.0 toabout 100 grams foam/gram crosslinking agent.

[0028] When a diacid halide is employed as crosslinked agent, an acidscavenger, e.g., triethylamine, can be also added to the crosslinkingreaction mixture to neutralize the hydrochloric acid that is formedduring the crosslinking reaction. The crosslinked foam is washed a fewtimes to remove any unreacted crosslinking agent, dried under vacuum andsterilized.

[0029] Subsequently, the crosslinked foam is mixed with a sterilizedaqueous solution which contains from about 0.5 to about 4.0% by weighthyaluronic acid, and preferably at about 2.0% by weight hyaluronic acid.Typically, the ratio of crosslinked foam to aqueous solution containinghyaluronic acid employed in the mixture ranges from about 0.2 to about1.0 gram crosslinked foam/ml hyaluronic acid solution. When thecrosslinked foam is crosslinked with diglycolyl chloride, generally, theaqueous solution containing hyaluronic acid which contains from about0.5 to about 2.0% by weight hyaluronic acid is mixed with thecrosslinked foam. The proportion of foam crosslinked with diglycolylchloride to the hyaluronic acid solution is preferably 0.2 gramscrosslinked hyaluronic acid foam/ml of a hyaluronic acid solutioncontaining 1.0% by weight hyaluronic acid.

[0030] The crosslinked hyaluronic acid foam and solution may be mixed byany convenient method. For example, the foam and solution may each beplaced in a syringe coupled with a luer lock, and the contents of eachsyringe mixed by pushing the plungers back and forth. The resultinghydrogel anti-adhesion barrier can then be collected in one of thesyringes.

[0031] The anti-adhesion barrier is biodegradable and is thus reabsorbedover a period of time, obviating the need for subsequent surgery toremove the barrier. The rate of degradation of the anti-adhesion barriercan be controlled by increasing the degree of crosslinking.

[0032] In another aspect, the anti-adhesion barrier can be used toprevent or inhibit the formation of adhesions in an animal following anytype of surgery or trauma, by applying an effective amount of theanti-adhesion barrier described above to a wound site. The wound siterefers to a site of tissue that has been injured in any manner, e.g.,through surgery, contusion, abrasion, and so forth, and also refers totissues or organs that are adjacent to the injured tissue. For example,the barrier may be used to prevent or inhibit adhesions that form inrelation to intestinal surgery, e.g., bowel resection, hernia repair,etc., which may cause obstruction of the intestine. The barrier may alsoprevent or inhibit adhesions that form near a bone fracture site whichmay reduce or hinder the normal movement of the area of repair byrestricting the natural movement of tendons over adjacent bone.

[0033] An effective amount of an anti-adhesion barrier is that amountrequired to prevent or inhibit adhesions. Preferably, the amount shouldbe sufficient to coat the entire wound site. An additional amount mayalso be applied to body tissues or organs adjacent to the wound site.The effective amount can be determined readily by one skilled in theart.

[0034] In one embodiment, as described above, the crosslinked foam andhyaluronic acid solution are mixed outside of the body to form ahyaluronic acid anti-adhesion barrier. The barrier is then applied tothe wound site by any convenient mode. Preferably, the anti-adhesionbarrier is applied directly to the wound site by injection through aneedleless syringe as set forth in Example 2.

[0035] In another aspect, the barrier may be used to promote healing ofa wound by applying an effective amount of the barrier described aboveto the wound. An additional amount may also be applied to the bodytissue adjacent to the wound. The effective amount can be determinedreadily by one skilled in the art.

[0036] The barrier is administered to the wound by any convenient mode,e.g., by applying the gel directly to the wound, or the gel may becombined with a wound dressing or bandage that is applied to the wound,or any other convenient mode.

[0037] The anti-adhesion barrier used to prevent or inhibit adhesions,or promote healing of a wound as described above may be included incompositions with a pharmaceutically acceptable carrier, e.g., water ora nonaqueous solvent, and/or at least one medicinal agent. Suitablemedicinal agents may be included in the compositions by adding themedicinal agent to the hyaluronic acid solution prior to mixing thesolution with the crosslinked foam to form the anti-adhesion barrier, orafter formation of the anti-adhesion barrier.

[0038] The term “medicinal agent”, as used herein, is meant to beinterpreted broadly and includes any substance or mixture of substanceswhich may have any clinical use in medicine. Thus, medicinal agentsinclude drugs, enzymes, proteins, peptides, glycoproteins, or diagnosticagents such as releasable dyes which may have no biological activity perse.

[0039] Examples of classes of medicinal agents that can be used includeantimicrobials, analgesics, antipyretics, anesthetics, antiepileptics,antihistamines, anti-inflammatories, anti-clotting agents,cardiovascular drug, diagnostic agents, sympathomimetics,cholinomimetics, anti-muscarinics, antispasmodics, hormones, growthfactors, muscle relaxants, adrenergic neuron blocks, anti-neoplastics,immunosuppressants, gastrointestinal drugs, diuretics, steroids andenzymes. It is also intended that combinations of medicinal agents canbe used.

[0040] By incorporating a medicinal agent(s) into the hyaluronic acidanti-adhesion barrier of this invention, focal delivery and applicationof a medicinal agent(s) to a wound is achieved. Focal application ofgrowth factors, anti-inflammatories immune system suppressant and/orantimicrobials by the anti-adhesion barrier is an ideal drug deliverysystem to speed healing of a wound or incision. Delivery of suitableanti-clotting agents in prevent fibroblast formation, thus augmentingthe effect of the physical barrier in preventing or inhibitingadhesions. The medicinal agent(s) diffuse from the hydrogel barrierand/or are released as the barrier is biodegraded and absorbed.

[0041] In another aspect, a two-part kit is provided which includes afirst part including a freeze-dried crosslinked hyaluronic acid foam,and a second part including a solution including hyaluronic acid. Thekit can also include at least one medicinal agent as described above,which may be included separately from the other components in the kit,or may be added to the solution including hyaluronic acid supplied inthe kit. The kit can also include any convenient means for mixing thecrosslinked hyaluronic acid foam with the solution including hyaluronicacid.

[0042] One example of a means for combining the crosslinked hyaluronicacid foam with the aqueous solution containing hyaluronic acid is amodified version of the syringe described in U.S. Pat. No. 3,682,174,which is incorporated herein by reference. With reference to FIG. 1, themodified syringe, i.e., needleless syringe, includes an outer casing 1of a tubular member having a bore 2, which hold the powdered substance3, i.e., hyaluronic acid foam. A second tubular member 4 holds theliquid 5, i.e., an aqueous solution containing hyaluronic acid. Thepowdered substance 3 is sealed off from the second tubular member usinga floating sealing member 6 and a sealing member 7. Thus, the powdercontained in the first member is completely separated from the secondmember. When an actuator (not shown in this figure) connected to the topportion of the syringe is pressed down, the plug 8 is displaced, whichin turn, places the liquid 5 under pressure. The sealing member 7becomes distorted, allowing the liquid to flow into the bore 2 of thefirst tubular member. The liquid mixes with the powder, with shaking ifrequired, to provide a freshly prepared suspension, i.e., a gelanti-adhesion barrier. The gel is held in place by a covering 9 which issecured to the bottom portion of the syringe. The covering is removed toallow the gel to be dispersed and administered to the desired site.

[0043] In a second example, the crosslinked hyaluronic acid foam andhyaluronic acid solution are mixed by placing each in a separateneedleless syringe which is coupled to a luer lock as described above,followed by mixing of the contents. With reference to FIG. 2A, thesyringe 1 contains an aqueous solution of hyaluronic acid 2 and syringe3 contains a crosslinked hyaluronic acid foam 4. The luer lock 5 is usedfor coupling syringe 1 to syringe 3. FIG. 2B shows the coupling ofsyringe 1 to syringe 3 using the luer lock 5. The aqueous solution ofhyaluronic acid 2 and the crosslinked hyaluronic acid foam 4 are mixedby pushing the plungers 6 and 7 back and forth to form the gelanti-adhesion barrier. Before applying the gel to a wound site, it iscollected in either syringe 1 or syringe 3.

[0044] In order that those skilled in the art may be better able topractice the present invention, the following examples are given asillustrations of the preparation of an anti-adhesion barrier, andmethods of use thereof in accordance with the present disclosure. Itshould be noted that the invention is not limited to the specificdetails embodied in the examples.

EXAMPLE 1

[0045] A. Preparation of Hyaluronic Acid Foam

[0046] Cosmetic grade hyaluronic acid (5.0 g) (Genzyme Corp., Cambridge,Mass.) was dissolved in distilled water (1000 ml). The hyaluronic acidsolution (0.5%) was stirred at room temperature for approximately 12hours. Hyaluronic acid (125 ml) solution was added to a 250 ml flask,followed by rapidly freezing the solution at about −94° C., by immersingthe flask in an acetone/dry ice bath. The frozen solution was thenplaced in a lyophilizer, at a vacuum of less than 1 torr for 3 to 4days, to form a hyaluronic acid foam.

[0047] B. Preparation of Diglycolyl Chloride Solution

[0048] A 3 necked round-bottomed flask (2 liter) fitted with a 300 mladdition funnel and a condenser with hydrogen flow was charged withdiglycolic acid (100 g) and toluene (1000 ml), followed by addition ofN,N-dimethylformamide (1-2 ml), a catalyst. From the addition funnel,thionyl chloride (275 ml) was added dropwise to the round bottomed flaskwhile stirring. The mixture was heated to 50° C., and then kept heatedovernight. The reaction was then allowed to continue at room temperaturefor two days. The toluene and thionyl chloride were distilled off at 50°C. under a vacuum, followed by heating the mixture at 80° C. under avacuum. The diglycolyl chloride was collected in a receiving flask. Upondistillation, the diglycolyl chloride (125 g) turned into a pinkishliquid. Nuclear Magnetic Resonance confirmed that all of the toluene wasdistilled off.

[0049] C. Preparation of Crosslinked Hyaluronic Acid Foam

[0050] Hyaluronic acid foam (0.325 g) prepared in Step A was immersed ina beaker containing diglycolyl chloride (100 g) which was prepared instep B. Triethylamine (1.0 ml) was added dropwise to the beaker toneutralize any hydrochloric acid that was generated during thecrosslinking reaction, and the mixture was stirred for 24 hours at roomtemperature. The crosslinked foam was then separated from diglycolylchloride using a sintered glass funnel. The collected crosslinked foamwas washed three times with 200 ml of isopropanol to remove unreacteddiglycolyl chloride, followed by a wash with 200 ml of ethanol. Thewashed crosslinked foam was then dried under vacuum at −40° C. for 24-48hours to yield approximately 0.3 grams of crosslinked hyaluronic acidfoam.

[0051] D. Formulation and Delivery of the Crosslinked Hyaluronic AcidGel

[0052] The crosslinked hyaluronic acid foam was sterilized by ethyleneoxide and a 1% hyaluronic acid solution was sterilized by steam for 120minutes at 121° C. Under aseptic conditions, hyaluronic acid foam (0.2g) was packed into a sterile 3 cc syringe, and hyaluronic acid solution(1 ml) was added to another syringe. Both syringes were uncapped,coupled-with a luer lock, and the contents of each syringe were mixed bypushing the plungers back and forth. The gel was collected in one of thesyringes, and then delivered to the surgical site.

EXAMPLE 2

[0053] Crosslinked Hyaluronic Acid Gel Employed as Anti-Adhesion Barrierin Rats

[0054] The ability of the crosslinked hyaluronic acid gel of Example 1to prevent or inhibit the formation of post-surgical adhesions wasexamined using a standardized rat abdominal adhesion model.

[0055] The test materials employed in the model were as follows:

[0056] Hyaluronic Acid Gel, 8 individually sealed packages, eachcontaining two syringes with the separate components for a single (orhalf) application of gel (mixed prior to surgery).

[0057] The wet and dry components of the hyaluronic acid gel kit weremixed prior to use using a syringe coupler to aerate and combine thecomponents as they were passed from one syringe to the other. The mixedgel was transferred to one of the syringes for application. In 3animals, the entire contents of the syringe was applied to the abdominalwall defect (AWD), completely coating it with a 5 mm margin beyond eachedge of the defect. In 5 animals only half of the volume of the gel wasapplied to the AWD of each animal. In all animals saline was drippedonto the exposed surface of the gel.

[0058] Female Sprague-Dawley rats (225-249 grams) were utilized in thisexperiments. Each animal received a 2×1 cm surgical defect on theperitoneal surface of their right abdominal wall, and a similar sizeddefect on their cecum. The animals were divided into two treatmentgroups. In the first group, the AWD was covered by a layer of hyaluronicacid gel. The animals in the second group served as controls with notreatment applied to either defect. All animals were sutured closed andbandaged.

[0059] The details of the model are described in Surgery 117: 663-9,1995, the contents of which are incorporated by reference herein. Inbrief, the animals were anesthetized with sodium pentobarbital (43 mg/kgintraperitoneal), their abdomens were shaved, and a 6 cm incision linewas marked on the skin overlying the linear alba on the ventral midline.The skin was prepped with iodophor solution, rinsed with 70% isopropylalcohol, and incised. With the muscle wall exposed, a 5 cm incision inthe muscle was made along the linear alba through to the peritonealcavity. The right abdominal wall was reflected, and a 2 cm×1 cm surfacewas denuded by removing the peritoneum and some associated musclefibers. The medial edge of this defect was located parallel to, and 1 cmlateral to the midline incision. The contents of the cecum were removedby milking the material away from the terminal end of the cecum.

[0060] A defect was created on the cecum by rubbing a moistened gauzepad on the surface of the cecum until a 2 cm×1 cm area of the serosalsheath covering the cecum was peeled away. Both the abdominal wall andthe cecum were lightly scraped with a #15 scalpel blade to promotepetechial bleeding, then exposed and allowed to air dry for 15 minutes.The non-defect areas of the abdominal wall and cecum were protected fromdrying by placing moist gauze over them during the drying period.

[0061] The prepared hyaluronic acid gel was quickly applied through the(needleless) syringe to the abdominal wall defect. An excess margin ofgel was extended beyond the defect area at least 5 mm. The abdominalwall was closed with a running 4/0 polypropylene suture, and the skinwas closed with a running 4/0 absorbable suture. The rats were bandagedand returned to their cages after anesthetic recovery.

[0062] The presence of adhesion formation between the cecum and theperitoneal wall was assessed 7 days following surgery. The animals wereeuthanized immediately prior to analysis. The skin and muscle layers ofthe abdomen were incised lateral and distal to the location of theoriginal defects. The resulting U-shaped flap was slowly lifted toreveal the adhesion, if present. After carefully noting and separatingany extraneous adhesions (i.e., retroperitoneal fat, bowel, omentum,etc.), two large silk suture loops were placed in the terminal end ofthe cecum. This permitted the cecum to be attached to a strain gaugemounted on the lead screw of a tensiometer. The caudal edge of theU-shaped flap was secured in a pin clamp such that the peritoneal wallwas approximately 40-45° from the horizontal. As the lead screw wasadvanced at 8.8 cm/min., the cecum was peeled off the peritoneal defect.The force required to remove the cecum was plotted against time on acalibrated x-y recorder. After the two defect surfaces were separated,the length and width of the peritoneal area involved in the adhesionwere measured with a caliper.

[0063] The following values were calculated for every animal with anadhesion: area of adhesion, percent of complete adhesion formation,maximum strength encountered during separation, maximum width ofadhesion, average strength of separation, work to separate, and anormalized work value. The normalized work value was calculated becausethe work to separate each adhesion is related to adhesion area.Therefore, each work value was normalized to the work that would havebeen required if the entire surface had been involved in the adhesion bydividing the actual work by the actual percentage of surface involved.

[0064] In order to calculate work and normalized work, the area of eachforce-time curve on the x-y recorder was measured with a planimeter,multiplied by a factor converting curve height to force in grams, anddivided by the curve length to obtain a value of average force in gramsfor each adhesion. The work to pull the adhesion was calculated usingthe formula W=F·d, where W=work, F=the average force, and d=the measuredlength of the peritoneal area involved in the adhesion. The normalizedwork was then calculated as the work, W, divided by the percentage ofcomplete adhesion formation. When reported, forces were converted fromgrams to Newtons (N) using the acceleration due to gravity. TABLE 1 Theresults are summarized in Table 1. Incidence of adhesion formation at 7days between an abdominal wall wound and a cecal defect in the ratfollowing with an absorbable hyaluronic acid (hyaluronic acid)-basedmaterial. INCIDENCE OF ADHESION FORMATION # of animals with adhesion/TREATMENT # of animals treated % NONE (CONTROLS) 11/11 100 HYALURONICacid GEL 1/3  33 (full dose) HYALURONIC ACID 1/5  20 GEL (half dose)

[0065] With hyaluronic acid gel, 2 out of 8 animals treated developedadhesions; one in the full-dose group and one in the half-dose group.One of these adhesions only involved 4% of the wound surface and wasconsidered to be an extremely minor adhesion (Table 1).

[0066] In contrast to the treated animals, all animals that did notreceive treatment developed adhesions (Table 1). All control animalsdeveloped strong 1:1 adhesions with a mean normalized work value of1.61+0.74 N−cm, a mean area of 1.29 cm² and a mean maximum strength 1.07N. These adhesions involved a mean of 42% of the wound area and requireda mean of 0.83 N cm to separate.

[0067] It will be understood that various modifications may be made tothe embodiments disclosed herein. Therefore, the above descriptionshould not be construed as limiting, but merely as exemplifications ofpreferred embodiments. Those skilled in the art will envision othermodifications within the scope and spirit of the claims appended hereto.

What is claimed is:
 1. A method of forming an anti-adhesion barriercomprising: freeze-drying a solution including hyaluronic acid to form afoam; reacting the foam with a crosslinking agent to form a crosslinkedfoam; and mixing the crosslinked foam with an aqueous solutioncontaining hyaluronic acid to form an anti-adhesion barrier.
 2. Themethod according to claim 1 wherein the concentration of the solutionincluding hyaluronic acid is from about 0.5 to about 4.0% by weighthyaluronic acid.
 3. The method according to claim 1 wherein during thefreeze-drying step the solution including hyaluronic acid is frozen at atemperature of about −94 to about −130° C.
 4. The method according toclaim 1 wherein the solution including hyaluronic acid is freeze-driedat less than about 50×10⁻³ torr and about −10° C.
 5. The methodaccording to claim 1 wherein the crosslinking agent is selected from thegroup consisting of di-epoxides, polyfunctional epoxides, diisocyanates,polyisocyanates, polyhydric alcohols, water-soluble carbodiimides,diaminoalkanes, diamines, polycarboxylic acids, diacid halides anddimethylol urea.
 6. The method according to claim 5 wherein thecrosslinking agent is a diacid halide.
 7. The method according to claim6 wherein the diacid halide is diglycolyl chloride.
 8. The methodaccording to claim 1 wherein a solution of crosslinking agent containsfrom about 10 to about 80 % by weight crosslinking agent.
 9. The methodaccording to claim 1 wherein the proportion of foam to crosslinkingagent is from about 3.0 to about 100 grams foam/gram of crosslinkingagent.
 10. The method according to claim 1 wherein the aqueous solutioncontaining hyaluronic acid solution contains from about 0.5 to about4.0% by weight hyaluronic acid.
 11. The method according to claim 1wherein the proportion of the crosslinked foam to the aqueous solutioncontaining hyaluronic acid is from about 0.2 to about 1.0 gramscrosslinked foam/ml aqueous solution containing hyaluronic acid.
 12. Ananti-adhesion barrier which is a gel produced by combining afreeze-dried crosslinked hyaluronic acid foam with an aqueous solutioncomprising hyaluronic acid.
 13. The anti-adhesion barrier according toclaim 12 wherein the crosslinked foam is crosslinked with a diacidhalide.
 14. The anti-adhesion barrier according to claim 13 wherein thediacid halide is diglycolyl chloride.
 15. A composition comprising anantiadhesion barrier according to claim 12, and a pharmaceuticallyacceptable carrier.
 16. The composition according to claim 15 furtherincluding a medicinal agent.
 17. An anti-adhesion barrier producedaccording to the method of claim
 1. 18. The anti-adhesion barrieraccording to claim 17 wherein the solution including hyaluronic acidcontains from about 0.5 to about 4.0% by weight hyaluronic acid.
 19. Theanti-adhesion barrier according to claim 17 wherein the crosslinkingagent is a diacid halide.
 20. The anti-adhesion barrier according toclaim 19 wherein the diacid halide is diglycolyl chloride.
 21. Acomposition comprising an antiadhesion barrier according to claim 17,and a pharmaceutically acceptable carrier.
 22. The composition accordingto claim 21 further including a medicinal agent.
 23. A method ofpreventing or inhibiting the formation of adhesions comprising applyingan effective amount of the anti-adhesion barrier according to claim 12to a wound site.
 24. The method according to claim 23 wherein thecrosslinked foam is crosslinked with diglycolyl chloride.
 25. A methodof promoting healing of a wound comprising applying an effective amountof the anti-adhesion barrier according to claim 12 to the wound.
 26. Atwo-part kit comprising a first part including a freeze-driedcrosslinked hyaluronic acid foam; and a second part including a solutionincluding hyaluronic acid.
 27. The kit according to claim 26 wherein thecrosslinked hyaluronic acid foam is crosslinked with a diacid halide.28. The kit according to claim 27 wherein the diacid halide isdiglycolyl chloride.
 29. The kit according to claim 26 further includingmeans for combining the crosslinked hyaluronic acid foam and thesolution including hyaluronic acid.
 30. The kit according to claim 26further including a medicinal agent.
 31. An anti-adhesion barriercomprising a hyaluronic acid foam, a crosslinking agent and an aqueoussolution containing hyaluronic acid.
 32. The anti-adhesion barrieraccording to claim 31 wherein the crosslinking agent is selected fromthe group consisting of di-epoxides, polyfunctional epoxides,diisocyanates, polyisocyanates, polyhydric alcohols, water-solublecarbodiimides, diaminoalkanes, diamines, polycarboxylic acids, diacidhalides and dimethylol urea.
 33. The anti-adhesion barrier according toclaim 32 wherein the crosslinking agent is a diacid halide.
 34. Theanti-adhesion barrier according to claim 33 wherein the diacid halide isdiglycolyl chloride.
 35. The anti-adhesion barrier according to claim 31wherein the solution containing hyaluronic acid contains from about 0.5to about 4.0% by weight hyaluronic acid.
 36. A composition comprising ananti-adhesion barrier according to claim 31, and a pharmaceuticallyacceptable carrier.
 37. The composition according to claim 36 furtherincluding a medicinal agent.